The BB Rat as a Model of Human Insulin‐Dependent Diabetes Mellitus

Barbara J. Whalen1, John P. Mordes1, Aldo A. Rossini1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.3
DOI:  10.1002/0471142735.im1503s19
Online Posting Date:  May, 2001
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Abstract

Use of the BioBreeding (BB) rat to model human insulin‐dependent diabetes mellitus (IDDM) is useful in that characteristics of diabetes in the BB rat closely parallel those observed in human IDDM. Diabetic animals can be biopsied, autopsied, and bred to study the genetic basis of IDDM. The genetic, immunological, and environmental components of the disease can all be investigated under controlled conditions. Two inbred lines of BB have been used in the majority of published studies using this model system; these rats are designated as diabetes prone (DP‐BB/Wor) and diabetes resistant (DR‐BB/Wor). This unit presents two protocols for the diagnosis and prevention of spontaneous IDDM in DP‐BB/Wor rats. Two alternate protocols are given for the induction of autoimmune disease in DR‐BB/Wor rats and for the adoptive transfer of autoimmunity into histocompatible athymic WAG nu/nu recipient rats. Support protocols are also given for diagnosis of insulitis, treatment of diabetic rats with insulin, and serological analysis of blood samples from sentinel rats to monitor the pathogen status of the colony.

     
 
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Table of Contents

  • Basic Protocol 1: Maintenance and Diagnosis of Spontaneous IDDM in Diabetes‐Prone BB Rats
  • Alternate Protocol 1: Induction of IDDM in Diabetes‐Resistant BB Rats
  • Alternate Protocol 2: Adoptive Transfer of IDDM
  • Basic Protocol 2: Prevention of IDDM in Diabetes‐Prone BB Rats
  • Support Protocol 1: Diagnosis of Insulitis
  • Support Protocol 2: Insulin Therapy for Diabetic Rats
  • Support Protocol 3: Serological Analysis of Blood Samples from Sentinel Rats
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Maintenance and Diagnosis of Spontaneous IDDM in Diabetes‐Prone BB Rats

  Materials
  • DP‐BB rats <50 days of age (available from Dennis Guberski; see )
  • Food (e.g., Purina), autoclaved
  • Drinking water: acidified tap or sterile water, adjusted to pH 3.5 to 4.0 with 1 N HCl
  • Clidox (Pharmacal Research Laboratories) disinfecting solution: 1 part activator, 1 part base, and 5 parts tap water, prepared fresh
  • Sentinel animals: DR‐BB or immunocompetent non‐BB rats
  • 0.3‐ml heparinized microcuvette capillary tubes (Sarstedt)
  • Glucose reagent strips (Clinistix, Miles Labs)
  • Microisolator housing (see unit 1.2), autoclaved, and if possible in a specific‐pathogen‐free (SPF) or viral‐antibody‐free (VAF) facility
  • Cages, autoclaved
  • Bedding, autoclaved
  • Water bottles, autoclaved
  • 100‐ to 300‐g scale
  • Calibrated instrumentation for measuring glucose concentration by the glucose oxidase method (e.g., Glucose Analyzer II, Beckman, or One Touch II, Lifescan)
  • Additional reagents and equipment for management of immunocompromised animals (unit 1.2), serological analysis of blood samples from sentinel rats (see protocol 7), tail vein blood collection (unit 1.7), diagnosis of insulitis (see protocol 5), and treatment of diabetic rats with insulin (see protocol 6)
NOTE: All materials taken into SPF or VAF animal rooms should be vacuum autoclaved, if possible. Nonautoclavable materials and the exposed surfaces of tubes, equipment, and other objects should be disinfected by spraying with Clidox disinfecting solution. Personnel handling the animals should wear sterile gowns or scrub suits, bonnets, masks, gloves, and shoe covers.

Alternate Protocol 1: Induction of IDDM in Diabetes‐Resistant BB Rats

  • Hybridoma DS4.23 cells (available from the Division of Diabetes, University of Massachusetts Medical School, Worcester, Mass.)
  • Complete RPMI medium ( appendix 2) supplemented with 2% (v/v) FBS, hybridoma serum supplement, or DR‐BB rat serum (RPMI‐2)
  • Four 4‐ to 6‐week‐old DR‐BB rats (available from Dennis Guberski; see )
  • 21‐ to 30‐day old DR‐BB rats of either sex (available from Dennis Guberski)
  • Polyinosinic:polycytidylic acid, sodium salt [poly(IC), Sigma], sterile
  • PBS ( appendix 2), sterile
  • Additional reagents and equipment for preparing MAb supernatants (unit 2.6),removal of lymphoid tissues (unit 1.9), immunofluorescence staining of single‐cells suspensions (unit 5.3), and screening and diagnosis of IDDM ( protocol 1)

Alternate Protocol 2: Adoptive Transfer of IDDM

  Materials
  • DR‐BB rats treated to induce IDDM (see protocol 2)
  • Anti‐RT6.1 MAb (e.g., supernatant from hybridoma DS4.23; see protocol 2)
  • WAG nu/nu RT1u rats of either sex, 6 to 12 weeks of age (VAF breeding stock available from the Division of Diabetes, University of Massachusetts Medical School, Worcester, Mass.)
  • Complete RPMI medium, HEPES‐buffered ( appendix 2)
  • Nylon cell strainer, sterile
  • Disposable hand towels, sterile
  • 1‐ml syringes
  • Glass beaker, sterile
  • Gauze, sterile
  • Additional reagents and equipment for euthanasia by CO 2 inhalation (unit 1.8), removing lymph nodes (unit 1.9), preparing single‐cell suspensions (unit 3.1), counting cells ( appendix 3A), and screening and diagnosis of diabetes (see protocol 1)
NOTE: All solutions and equipment coming into contact with cells should be sterile, and proper sterile technique should be used accordingly. Injections should be performed in a laminar flow hood in the animal room, with as much adherence to sterile technique as possible.

Basic Protocol 2: Prevention of IDDM in Diabetes‐Prone BB Rats

  Materials
  • 21‐ to 30‐day old DP‐BB rats of either sex (available from Dennis Guberski; see )
  • 8‐ to 12‐week old DR‐BB rats of either sex (available from Dennis Guberski)
  • Complete RPMI medium, HEPES‐buffered ( appendix 2)
  • Nylon cell strainer, sterile
  • 1‐ml syringes with 23‐G needles
  • Additional reagents and equiment for euthanasia (unit 1.8), removing the spleen (unit 1.9), preparing a single‐cell suspension (unit 3.1), counting cells ( appendix 3A), intraperitoneal injections (unit 1.6), and screening and diagnosis of diabetes (see protocol 1)
NOTE: All solutions and equipment coming into contact with cells should be sterile, and proper sterile technique should be used accordingly. Injections should be performed in a laminar flow hood in the animal room, with as much adherence to sterile technique as possible.

Support Protocol 1: Diagnosis of Insulitis

  Materials
  • DR‐BB or DP‐BB rat
  • recipeBouins fixative (see recipe)
  • 10% (v/v) buffered formalin (Baxter)
  • Dissecting instruments
  • Tissue‐processing histology cassettes (Fisher)
  • Histology pencil (Fisher)
  • Additional reagents and equipment for CO 2 euthanasia (unit 1.8)

Support Protocol 2: Insulin Therapy for Diabetic Rats

  Materials
  • Diabetic rats (see protocol 1 or protocol 2 or protocol 32)
  • Glucose reagent strips (Clinistix, Miles Labs)
  • U‐100 Ultralente insulin, bovine or porcine (Novo Nordisk Bioindustrials)
  • Insulin diluent (Novo Nordisk Bioindustrials)
  • Linplant sustained‐release bovine insulin implants and standard 12‐G trocar/stylet (Linshin Canada)
  • Ketone reagent strips (Ketostix, Miles Labs)
  • Lactated Ringers solution (Baxter), sterile
  • 0.5‐ml U‐100 insulin syringes, sterile
  • Additional reagents and equipment for anesthesia by methoxyflurane inhalation (unit 1.4) and subcutaneous injection (unit 1.6)

Support Protocol 3: Serological Analysis of Blood Samples from Sentinel Rats

  Materials
  • PBS ( appendix 2)
  • Additional reagents and equipment for anesthesia by methoxyflurane inhalation (unit 1.4), tail vein or orbital plexus blood collection (unit 1.7), and preparation of serum from blood (unit 2.4)
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Figures

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Literature Cited

Literature Cited
   Burstein, D., Mordes, J.P., Greiner, D.L., Stein, D., Nakamura, N., Handler, E.S., and Rossini, A.A. 1989. Prevention of diabetes in the BB/Wor rat by a single transfusion of spleen cells: Parameters that affect the degree of protection. Diabetes 38:24‐30.
   Crisá, L., Greiner, D.L., Mordes, J.P., Handler, E.S., Angelillo, M., Nakamura, N., and Rossini, A.A. 1990. Biochemical studies of RT6 alloantigens in BB/Wor and normal rats: Evidence for intact but unexpressed RT6a structural gene in diabetes‐prone BB rats. Diabetes 39:1279‐1288.
   Crisá, L., Mordes, J.P., and Rossini, A.A. 1992. Autoimmune diabetes mellitus in the BB rat. Diabetes Metab. Rev. 8:9‐37.
   Doukas, J., Mordes, J.P., Swymer, C., Niedzwiecki, D., Mason, R., Rozing, J., Rossini, A.A., and Greiner, D.L. 1994. Thymic epithelial defects and predisposition to autoimmune disease in BB rats. Am. J. Path. 145:1517‐1525.
   Dyrberg, T., Schwimmbeck, P.L., and Oldstone, M.B.A. 1988. Inhibition of diabetes in BB rats by virus infection. J. Clin. Invest. 81:928‐931.
   Edouard, P., Hiserodt, J.C., Plamondon, C., and Poussier, P. 1993. CD8+ T cells are required for adoptive transfer of the BB rat diabetic syndrome. Diabetes 42:390‐397.
   Greiner, D.L., Handler, E.S., Nakano, K., Mordes, J.P., and Rossini, A.A. 1986. Absence of the RT‐6 T cell subset in diabetes‐prone BB/W rats. J. Immunol. 136:148‐151.
   Greiner, D.L., Mordes, J.P., Handler, E.S., Angelillo, M., Nakamura, N., and Rossini, A.A. 1987. Depletion of RT6.1+ T lymphocytes induces diabetes in resistant BioBreeding/Worcester (BB/W) rats. J. Exp. Med. 166:461‐475.
   Greiner, D.L., Mordes, J.P., Angelillo, M., Handler, E.S., Mojcik, C.F., Nakamura, N., and Rossini, A.A. 1988. Role of regulatory RT6+ T‐cells in the pathogenesis of diabetes mellitus in BB/Wor rats. In Frontiers in Diabetes Research: Lessons from Animal Diabetes II (E. Shafir and A.E. Reynolds, eds.) pp. 58‐67. John Libby, London.
   Guberski, D.L., Thomas, V.A., Shek, W.R., Like, A.A., Handler, E.S., Rossini, A.A., Wallace, J.E., and Welsh, R.M. 1991. Induction of type 1 diabetes by Kilham's rat virus in diabetes resistant BB/Wor rats. Science 254:1010‐1013.
   Guberski, D.L., Manzi, S.M., and Like, A.A. 1992. Correction of lymphopenia by injection of diabetes‐resistant splenocytes prevents diabetes and improves breeding efficiency in diabetes‐prone BB/Wor rats. Contemp. Top. Lab. Animal Sci. 31:13.
   Jacoby, R.O. and Ball‐Goodrich, L.J. 1995. Parvovirus infections of mice and rats. Semin. Virol. 6:329‐337.
   Joseph, S., Diamond, A.G., Smith, W., Baird, J.D., and Butcher, G.W. 1993. BB‐DR/Edinburgh: A lymphopenic, nondiabetic subline of BB rats. Immunology 78:318‐328.
   McKeever, U., Mordes, J.P., Greiner, D.L., Appel, M.C., Rozing, J., Handler, E.S., and Rossini, A.A. 1990. Adoptive transfer of autoimmune diabetes and thyroiditis to athymic rats. Proc. Natl. Acad. Sci. U.S.A. 87:7718‐7722.
   Metroz‐Dayer, M.‐D., Mouland, A., Brideau, C., Duhamel, D., and Poussier, P. 1990. Adoptive transfer of diabetes in BB rats induced by CD4 T lymphocytes. Diabetes 39:928‐932.
   Mordes, J.P., Desemone, J., and Rossini, A.A. 1987. The BB rat. Diabetes Metab. Rev. 3:725‐750.
   Nakhooda, A.F., Like, A.A., Chappel, C.I., Murray, F.T., and Marliss, E.B. 1977. The spontaneously diabetic Wistar rat. Metabolic and morphologic studies. Diabetes 26:100‐112.
   Prins, J.‐B., Herberg, L., Den Bieman, M., and Van Zutphen, B.F.M. 1991. Genetic characterization and interrelationship of inbred lines of diabetes‐prone and not diabetes‐prone BB rats. In Frontiers in Diabetes Research: Lessons from Animal Diabetes III (E. Shafrir, ed.) pp. 19‐24. Smith‐Gordon, London.
   Rozing, J., Coolen, C., Tielen, F.J., Weegenaar, J., Schuurman, H.‐J., Greiner, D.L., and Rossini, A.A. 1989. Defects in the thymic epithelial stroma of diabetes‐prone BB rats. Thymus 140:125‐135.
   Rossini, A.A., Mordes, J.P., Handler, E.S., and Greiner, D.L. 1995. Human autoimmune diabetes mellitus: Lessons from BB rats and NOD mice—Caveat emptor. Clin. Immunol. Immunopathol. 74:2‐9.
   Thomas, V.A., Woda, B.A., Handler, E.S., Greiner, D.L., Mordes, J.P., and Rossini, A.A. 1991. Exposure to viral pathogens alters the expression of diabetes in BB/Wor rats. Diabetes 40:255‐258.
   Whalen, B.J., Greiner, D.L., Mordes, J.P., and Rossini, A.A. 1994. Adoptive transfer of autoimmune diabetes mellitus to athymic rats: Synergy of CD4+ and CD8+ T cells and prevention by RT6+ T cells. 1994. J. Autoimmunity 7:819‐831.
   Whalen, B.J., Rossini, A.A., Mordes, J.P., and Greiner, D.L. 1995. DR‐BB rat thymus contains thymocyte populations predisposed to autoreactivity. Diabetes 44:963‐967.
   Zipris, D., Greiner, D.L., Malkani, S., Whalen, B., Mordes, J.P., and Rossini, A.A. 1996. Cytokine gene expression in islets and thyroids of BB rats. Interferon‐γ and IL‐12p40 mRNA increase with age in both diabetic and insulin‐treated nondiabetic BB rats. J. Immunol. 156:1315‐1321.
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